Evolving genes through directed evolution procedures is a way of generating novel nucleic acids sequences that encode proteins that can perform desired functions quickly and efficiently. One method of directed evolution is error-prone PCR, in which a DNA Polymerase incorporates random single-base sequence errors into a product. (Leung et al., Technique (1989) 1, 11–15) Another technique is site directed mutagenesis, in which one or more particular amino acids in a gene are substituted for a different amino acid. Other methods utilize agents that induce random mutation, such as the addition of compounds such as nitrosoguanidine (NTG) to the culture media of a population of organisms (for example, see Nestmann E R Mutat Res 1975 June;28(3):323–30). The resulting nucleic acid sequences and genomic sequences created using these procedures comprise a library of sequences with differing abilities to perform a desired function, and the performance of that function is then assayed for and novel and desirable sequences are identified.
Assay procedures for desired functions may be employed to test the aforementioned nucleic acid sequences for a desired function. For instance, an enzyme that functions best at pH 5.0 may be mutagenized and the resulting population of sequences is then assayed for the production of the product of the enzymatic reaction at pH 7.0. Alternatively, sequences encoding antibodies may be mutagenized and then assayed for the ability to bind a particular molecule with an affinity that is stronger or weaker than the original antibody sequence.
The assay system for directed evolution procedures may be based on selection or screening, or both. In a typical selection protocol, organisms that are transformed with or contain mutagenized sequences are put through a procedure in which the ability to perform a given function is coupled with the ability to survive the conditions that the transformed organisms are subjected to. For instance, an enzyme that has potential bioremediation functions may be mutagenized and an organism is transformed with the library. Organisms that can detoxify a toxic compound using a mutagenized enzyme under conditions in which the toxic compound is supplied at a concentration that would otherwise kill the organism are selected for. In screening procedures, organisms that cannot perform the desired function survive, but do not identify mutagenized versions of genes that are desirable. For instance, an enzyme that cleaves a substrate that exhibits a first fluorescence emission wavelength spectrum when intact and a measurably distinct second fluorescence emission wavelength spectrum after catalysis may be screened for when the transformed organisms are subjected to specific fluorescent excitation wavelengths of light and the emission is monitored at two or more wavelengths. Enzymes that cleave the substrate at an enhanced rate may be identified.
Directed evolution is therefore a useful method through which genes may be adapted to perform desirable functions.